During physiologic coagulation, the complex of tissue factor (TF) and factor VIIa (FVIIa) activates FX to FXa and FIX to FIXa. In this process, the gamma-carboxyglutamic acid (Gla) domain and the epidermal growth factor- like domain 1 (EGF1) of FX and that of FIX play important roles in interactions with TF/FVIIa complex. This activation process is regulated by tissue factor pathway inhibitor (TFPI), which consists of three Kunitz domains; the first domain (K1) binds to FVIIa and the second domain (K2) binds to FXa (but not to FIXa). Thus, formation of the quaternary TF:VIIa:Xa:TFPI inhibitory complex effectively shuts down the TF-induced FX activation. However, uninhibited TF/VIIa continues to activate FIX for persistent coagulation. The molecular recognitions involved in the activations of FX and FIX by TF/VIIa and in the formation of the inhibitory complex (TF:VIIa:Xa:TFPI) are poorly understood. In this application, we propose crystallography studies to delineate the molecular interactions involved in binding of Gla and EGF1 domains of Xa and IXa to TF/FVIIa, and in binding of FVIIa to the K1 domain and of FXa to the K2 domain of TFPI. We will use two-domain TFPI (TFPI161) that binds to FXa and to FVIIa. Wild-type FXa after binding to TFPI via its active site serine slowly cleaves TFPI. Thus we will use FXa in which its active Ser195 is mutated to Ala (XaS195A). XaS195A binds to TFPI very tightly. To understand the interaction of Gla and EGF1 domains of FIXa with VIIa/sTF, we will use a hybrid XaS195A in which its Gla and EGF1 domains are replaced from FIX, termed XaS195A-IXGE. Two specific aims are: Aim 1: Crystallization and Structure Determination of (XaS195A)-VIIa-sTF-TFPI161 Quaternary Complex. For these studies, we have successfully purified the quaternary complex in stable form and obtained crystals that diffract to 3.5 A. We will continue our efforts to obtain crystals that diffact to high resolution for determining the molecular interactions in these proteins at the atomic level Aim 2: Crystallization and structure determination of (XaS195A-IXGE)-VIIa-sTF-TFPI161 quaternary complex. For these studies, we have also expressed and isolated each protein, and have successfully purified the quaternary complex in stable form. Initial crystallization trials are in progress. We realize that this is an ambitious proposal and involves high risk. However, determining the structure of the proposed quaternary complexes is the only way to gain such knowledge. Moreover, successful completion of this project will advance the filed in a highly significant way. The knowledge gained from the proposed studies could aid in the development of new generation of antithrombotics and antihemophilic agents.